Lanthanide complexes comprising at least two betaine groups, which can be used as luminescent markers

ABSTRACT

The present invention relates to luminescent lanthanide complexes including a chelating agent, formed with a macrocycle or a set of ligands, complexing a lanthanide ion Ln3+, wherein the chelating agent is substituted with at least two groups. The invention is most particularly applied to lanthanide complexes, the chelating agent of which is formed the with three ligands integrating a 2,6 pyridine-di(carboxylic acid) or is formed with a macrocycle having a 1,4,7 triazacyclononane structure.

This application is a 371 of PCT/FR2014/050813, filed on Apr. 3, 2014,which claims priority to French Application No. 1353037, filed Apr. 4,2013.

The present invention relates to the technical field of lanthanidecomplexes. In particular, the present invention relates to lanthanidecomplexes comprising at least two betaine groups on their organicportion, giving them interesting properties in terms of solubility inwater and in biological media, and allows limitation, or evensuppression of non-specific adhesion phenomena with biomolecules(proteins . . . ) or lipophilic portions of cells, notably allowingtheir use in biological applications.

Certain lanthanide complexes have remarkable spectroscopic properties(fine emission bands characteristic of a given metal and with a longlifetime) and are, consequently, compounds with a very strong potentialfor applications in biological imaging (S. V. Eliseeva, J.-C. G. Bünzli,Chem. Soc. Rev. 2010, 39, 189). These luminescent compounds may be usedalone for applications in imaging by mono- or bi-photon fluorescencemicroscopy, or else in conjunction with a suitable fluorophore forcarrying out FRET (Förster/Fluorescence Resonant Energy Transfer)experiments. In the latter case, the lanthanide complexes are generallyin the form of a conjugate complex with a biomolecule. Both of thesetechniques may optionally be resolved in time, by the long lifetime oflanthanides, which is an important advantage for improving detectionwhile getting rid of parasitic fluorescence signals with a shortlifetime (C. P. Montgomery B. S. Murray, E. J. New, R. Pal, D. Parker,Acc. Chem. Res. 2009, 42, 925. E. G. Moore, A. P. S. Samuel, K. N.Raymond, Acc. Chem. Res. 2009, 42, 542.). Many other luminescentlanthanide compounds have been described in the literature for suchapplications (M. Latva, H. Takalo, V.-M. Mukkala, C. Matachescu, J. C.Rodríguez-Ubis, J. Kankare, J. Lumin., 1997 75 149-169; A. Picot, A.D'Aléo, P. L. Baldeck, A. Grichine, A. Duperray, C. Andraud, O. Maury,J. Am. Chem. Soc. 2008, 130, 1532) and some of them are marketed asderivatives of DTPA (diethylene triamine penta acid, U.S. Pat. No.5,622,821), compounds based on pyridine or poly-pyridine (U.S. Pat. No.4,920,195; U.S. Pat. No. 4,761,481; U.S. Pat. No. 5,216,134; U.S. Pat.No. 4,859,777; U.S. Pat. No. 5,202,423; U.S. Pat. No. 5,324,825), orfurther macrocyclic cryptates (EP 0 180 492; EP 0 321353; EP 0 601 113;WO 2001/96877; WO 2008/063721).

In the past, lanthanide complexes had a simple structure with not verymuch carbon, were generally charged, and consequently were thereforesoluble in water. With the developments during these recent years,functionalized complexes were developed for optimizing theirspectroscopic properties, displacing their absorption towards visiblelight and optimizing their bi-photon absorption. Increasingly,functional antennas generally conjugate and/or aromatic including alarge number of carbons are introduced, which leads to lipophiliccompounds, therefore not soluble in water (C. Andraud, O. Maury Eur. J.Inorg. Chem. 2009, 4357-4371).

In order to increase hydrosolubilization of lanthanide complexes,different approaches were developed. One approach consists ofintroducing polyethylene glycol (PEG) groups as hydrosolubilizinggroups. A. Picot, A. D'Aléo, P. L. Baldeck, A. Grichine, A. Duperray, C.Andraud and O. Maury, in J. Am. Chem. Soc., 2008, 130, 1532-153.3 usedthis approach in the case of lanthanide complexes for which thecomplexation is ensured by three ligands integrating a2,6-pyridine-carboxylic diacid. The introduction of PEG groups is alsocontemplated in C. R. Chim., 2010, 13, 681-690, Inorg. Chem., 2011, 50,4987-4999 and Angew. Chem. In. Ed. 2012, 51, 6622-6625.

Patent application WO 2013/011236, relating to strongly luminescentlanthanide compounds, for which certain are based on a1,4,7-triazacyclononane ring (TACN) substituted with chromophores basedon conjugate pyridine, also use PEG groups. The described complexes arevery stable in water and the presence of PEG groups ensures their goodsolubility.

These functional macrocyclic ligands notably lead to the formation ofeuropium complexes which have very high brightness at 337 nm (thebrightness is defined by the product of the quantum yield by theabsorption at the wavelength of interest, here 337 nm), 337 nmcorresponding to the excitation wavelength of the nitrogen laser usedfor commercial applications (J. W. Walton, A. Bourdolle, S. J. Butler,M. Soulier, M. Delbianco, B. K. McMahon, R. Pal, H. Puschmann, J. M.Zwier, L. Lamarque, O. Maury, C. Andraud and D. Parker Chem. Commun.2013, 49, 1600-1602).

These compounds may also be provided with a reactive function, so as tobe optionally conjugate by a covalent bond to a molecule of interest,such as a biomolecule. In this case, the compounds have the end purposeof being bound covalently to a biomolecule, with view to conducting FRETexperiments (HTRF® applications marketed by CISBIO BIOASSAYS).

However, these compounds have two major drawbacks:

1) Within the scope of their research work, the inventors observed thatthese complexes are adsorbed in a non-specific way on biomolecules,which complicates the preparation of conjugate biomolecules. Thisphenomenon is also found in imaging experiments by mono- or bi-photonmicroscopy conducted on set cells. In this case, specific accumulationof these compounds in the lipophilic membranes and/or organelles isascertained. The inventors of the present invention associated thisdrawback with the presence of PEG groups.

2) Further, these compounds are prepared via a not very polyvalent, longand with modest efficiency convergent synthesis route.

Other solutions propose solubilization of the lanthanide complexes byintroducing anionic charged groups. In Inorg. Chem. 2009, 48(9),4207-4218, hydrosolubilization is ensured by —SO₃ groups, whereas inInorg. Chem., 2009, 48, 4601-4603, the hydrosolubilization is ensured by—CO₃ groups. In these examples, the introduction of anionic groups givesthe possibility of ensuring good hydrosolubilization, the problem of thenon-specific adsorption is not tackled and no example describes theirbehavior in the presence of bioconjugates.

The present invention proposes provision of novel fluorescent lanthanidecomplexes which have satisfactory solubility in water and therefore inphysiological and biological media, but they do not have the drawback ofthe previous solutions, and notably of those using PEG groups, for whichthe inventors ascertained a tendency to adhere to biomolecules, to cellmembranes and more generally to hydrophobic portions present in a largenumber in biomolecules and biological media.

In this context, the invention relates to luminescent lanthanidecomplexes including a chelating agent, formed with a macrocycle or a setof ligands, complexing a lanthanide ion Ln³⁺, wherein the chelatingagent is substituted with at least two betaine groups.

As an example of a lanthanide ion Ln³⁺, mention may be made of Eu³⁺,Sm³⁺, Tb³⁺ or Dy³⁺.

Within the scope of the invention, the solubilization of lanthanidecomplexes in an aqueous medium is ensured by zwitterionichydrosolubilizing groups of the betaine type.

The present invention proposes a functionalization of lanthanidecomplexes with two betaine groups or more. This functionalization givesthe possibility of solubilizing the lanthanide complexes in water andbiological media and makes it possible to avoid non-specific adhesionphenomena with biomolecules and organelles. This functionalization maybe applied to all types of lanthanide complexes. Further, within thescope of the invention, it was demonstrated that the presence of betainegroups did not affect the luminescence properties of the complexes.

Admittedly, betaines, in a very large number, had already been used forfunctionalizing nano-objects such as paramagnetic nanoparticles orquantum dots (H. Wei, N. Insin, J. Lee, H.-S. Han, J. M. Cordero, WenhaoLiu, M. G. Bawendi Nano Lett. 2012, 12, 22-25; E. Muro, T. Pons, N.Lequeux, A. Fragola, N. Sanson, Z. Lenkei, B. Dubertret J. Am. Chem. Soc2010, 132, 4556-4557). However, in this type of polyfunctionalnano-objects, it is impossible to determine whether the limitation ofthe non-specific adhesion phenomena observed might stem from the actualnature of the betaine group or from the simultaneous presence of a verylarge number of these functions (a so-called multivalence phenomenon).Thus, according to present knowledge, it was totally impossible topredict the behavior of small molecules, such as lanthanide complexes,functionalized with betaine groups. In the literature, betaine groupshave also been introduced on organic chromophores (cyanines, bodipy) formaking them soluble in water, but nothing is described as regards apossible property of non-adhesion. The non-adhesion property obtainedfor molecular compounds, like in the case within the scope of theinvention, was therefore by no means predictable.

A betaine group is defined as a zwitterionic group wherein the atombearing the positive charge does not bear any hydrogen atom and is notadjacent to the atom bearing the negative charge. Within the scope ofthe invention, by betaine is for example meant the zwitterionic groupsassociating an ammonium cation or aromatic iminium, generallypyridinium, imidazolium, and an anionic group of the sulfonate,phosphonate or carboxylate type, preferably sulfonate. The cation andthe anion are spaced apart by at least one CH₂ ring member, andpreferably by a bivalent alkyl chain (also-called alkylene) comprisingfrom 1 to 4, or even from 1 to 6 carbon atoms. As an example of abetaine group, mention may be made of the groups of formula:

with R representing an alkyl group from 1 to 6 carbon atoms, andpreferably a methyl or ethyl, and q being equal to 1, 2, 3, 4, 5 or 6,and preferably equal to 1 or 2. The group —N(CH₃)₂ ⁺—(CH₂)₃—SO₃ ⁻ ispreferred.

Advantageously, the lanthanide complexes according to the inventioninclude a chelating agent substituted with at most 12 betaine groups,for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 betaine groups.

Moreover, the complexes according to the invention may also be providedwith a reactive function, in order to allow their conjugation through acovalent bond to a molecule of interest, such as a biomolecule. Byreactive function, is meant a function allowing covalent grafting on areactive function present on a biomolecule (amine, alcohol, thiol,carboxylic acid, unsaturated functions . . . ). The various functionsallowing such bioconjugation are well known to one skilled in the artand for example are described in Bioconjugate Techniques, G. T.Hermanson, Academic Press, 1996, 137-166. Preferably, this reactivefunction will be an amine function, an activated ester, an azido group.In particular, the lanthanide complexes according to the invention willinclude one chelating agent substituted with at least one, and inparticular a single one, reactive function allowing its coupling througha covalent bond with a biomolecule, said reactive function beingpreferably selected among —COOH, —NH₂, an acrylamide, an activatedamine, an activated ester, an aldehyde, an alkyl halide, an anhydride,an aniline, an azide, an aziridine, a carboxylic acid, a diazoalkane, ahaloacetamide, a halotriazine, a hydrazine, an imido ester, anisocyanate, an isothiocyanate, a maleimide, a sulfonyl halide, a thiol,a ketone, an amine, an acid halide, a hydroxysuccinimidyl ester, asuccinimidyl ester, a hydroxysulfosuccinimidyl ester, anazidonitrophenyl, an azidophenyl, a 3-(2-pyridyl-dithio)-propionamide, aglyoxal, a triazine, an acetylenic group, and groups of formula:

wherein w is an integer belonging to the range from 0 to 8 and v isequal to 0 or 1, and Ar is a heterocycle with 5 or 6 members, eithersaturated or unsaturated, comprising from 1 to 3 heteroatoms, optionallysubstituted with a halogen atom;

the reactive functions selected from —COOH, —NH₂, succinimidyl esters,haloacetamides, azides, hydrazines, isocyanates and maleimides beingpreferred.

By biomolecules, are meant molecules of biological interest for which itmay be advantageous to mark them by means of a luminescent complex, forexample, proteins, peptides, antibodies, antigens, DNA strands, biotin,streptavidin. Proteins are biomolecules which will most often be boundto the complexes according to the invention.

Examples of lanthanide complexes according to the invention include achelating agent formed with three ligands integrating a2,6-pyridine-dicarboxylic acid group or formed with a macrocycle with a1,4,7-triazacyclononane structure.

In particular, the invention relates to lanthanide complexes selectedfrom the lanthanide complexes of formula (IV):

wherein:

-   -   Ln is a lanthanide, notably Eu, Sm, Tb or Dy,    -   Z represents —C— or —PR₃—,    -   R₃ represents a phenyl, benzyl, methyl, ethyl, propyl, n-butyl,        sec-butyl, iso-butyl or tert-butyl group and preferably a phenyl        or methyl group,    -   Chrom1, Chrom2 and Chrom3, either identical or different, are        selected from the groups:

wherein:

-   -   L₁ represents a direct bond, —C═C— or —C≡C—,    -   Ar₁ represents an aromatic group selected from phenyl,        thiophenyl, furanyl, pyrrolyl, imidazolyl or triazolyl groups,        substituted with s groups R₀ either identical or different,    -   s is equal to 1, 2 or 3, and    -   R₀ is selected among alkyl groups comprising from 1 to 10, and        preferably from 1 to 6 carbon atoms; the alkyl groups comprising        from 1 to 10 carbon atoms bearing at least one betaine function        and/or a reactive function; and electron donor groups, notably O        donors, S donors, NHCO donors, SCO donors, NHCS donors, and SCS        donors, said electron donor groups may either bear or not one or        several betaine groups, and/or a reactive function, it being        understood that when all the groups Ar1 represent a phenyl        group, at least one of these phenyl groups, preferably two or        even three of these groups, is (are) substituted with at least        one group R₀ including an electron donor group;

characterized in that at least two of the Chrom1, Chrom2 and Chrom3groups are substituted with at least one group R₀ bearing at least onebetaine group.

Preferably, in the complexes of formula (IV), Chrom1, Chrom2 and Chrom3are defined as follows:

-   -   a. either Chrom1, Chrom2 and Chrom3, either identical or        different, are selected from the groups:

-   -    with Ar1 as defined for the complexes of formula (IV), (which        corresponds to the case when L₁ represents a direct or covalent        bond),    -   b. or Chrom1, Chrom2 and Chrom3, either identical or different,        are selected from groups:

-   -    with Ar1 which represents a phenyl, thiophenyl, furanyl,        pyrrolyl or imidazolyl group substituted with s groups R₀ either        identical or different, s and R₀ being as defined for the        complexes of formula (IV).

In the complexes of formula (IV) according to the invention, the R₀,substituents, either identical or different, are, for example, selectedamong:

-   -   -L₂-Alk, -L₂-L₃-Q₁ and -L₂-L₃-Q₂;    -   the NHCO donor groups are selected from: —NHCO(OAlk),        —NHCO(NHAlk), —NHCO(NAlk1Alk2), —NHCO(SAlk),    -   the SCO donor groups are selected from: —SCO(OAlk), —SCO(NHAlk),        —SCO(NAlk1Alk2), —SCO(SAlk),    -   the NHCS donor groups are selected from: —NHCS(OAlk),        —NHCS(NHAlk), —NHCS(NAlk1Alk2), and    -   the SCS donor groups are selected from: —SCS(OAlk), —SCS(NHAlk),        —SCS(NAlk1Alk2), —SCS(SAlk),    -   Alk, Alk1 and Alk2, either identical or different, are alkyl        groups comprising from 1 to 10 carbon atoms, optionally        substituted with at least one betaine group,    -   Q₁ represents a betaine group or a branched group bearing at        least two betaine groups,    -   L₂ is a direct bond, —O—, —S—, —NHCO—, —SCO—, —NHCS— or —SCS—    -   L₃ is a bond arm, and    -   Q₂ is a reactive group which may allow the covalent bond with a        molecule of interest to be marked,

it being understood that at least two of the present R₀ substituentsbear at least one betaine group, so that at least two of the groupsChrom1, Chrom2 and Chrom3 are substituted with at least one R₀ groupbearing at least one betaine group.

As an example of a branched group bearing at least two betaine groups,mention may be made of the groups of the following formula:

with u which is equal to 1, 2, 3, 4, 5 or 6.

In the complexes of formula (IV) according to the invention, it ispossible that Chrom1=Chrom2=Chrom3 or preferably that Chrom1=Chrom2 andare substituted with at least one group R₀ bearing at least one betainegroup and that Chrom3 is substituted with at least one group R₀ bearinga function -L₂-L₃-Q₂, as defined earlier, with preferably Q₂ whichrepresents a group selected from —COOH, —NH₂, an acrylamide, anactivated amine, an activated ester, an aldehyde, an alkyl halide, ananhydride, an aniline, an azide, an aziridine, a carboxylic acid, adiazoalkane, a haloacetamide, a halotriazine, a hydrazine, an imidoester, an isocyanate, an isothiocyanate, a maleimide, a sulfonyl halide,a thiol, a ketone, an amine, an acid halide, a hydroxysuccinimidylester, a succinimidyl ester, a hydroxysulfosuccinimidyl ester, anazidonitrophenyl, an azidophenyl, a 3-(2-pyridyldithio)-propionamide, aglyoxal, a triazine, an acetylenic group, and the groups of formula:

wherein w is an integer belonging to the range from 0 to 8 and v isequal to 0 or 1, and Ar is a heterocycle with 5 or 6 members, eithersaturated or unsaturated, comprising from 1 to 3 heteroatoms, optionallysubstituted with a halogen atom;Q₂ is preferably selected from —COOH, —NH₂, succinimidyl esters,haloacetamides, hydrazines, isocyanates and maleimides being preferred.

The complexes of formula (IV) according to the invention, wherein L₁represents a direct bond or —C≡C—, and the groups Ar1, either identicalor different, each represent a phenyl group, substituted with s R₀groups either identical or different, with s and R₀ being as defined forthe complexes of formula (IV), are examples of complexes according tothe invention.

The complexes of formula (IV) according to the invention wherein s isequal to 1 for all the Ar1 groups are simpler to synthesize and will bepreferred.

As an example of complexes of formula (IV) according to the invention,mention may be made of those for which all the Ar1 groups, eitheridentical or different, represent a phenyl group selected from thegroups:

with R₀ as defined for the complexes of formula (IV).

The invention also relates to the lanthanide complexes of formula (III):

wherein:

-   -   Ln is a lanthanide, notably Eu, Sm, Tb or Dy,    -   Chrom4 is selected from the groups:

wherein:

-   -   L₁ represents a direct bond, —C═C— or —C≡C—,    -   Ar2 represents an aromatic group selected from phenyl,        thiophenyl, furanyl, pyrrolyl, imidazolyl or triazolyl groups,        said groups Ar2 being substituted with s groups R₁ either        identical or different,    -   s is equal to 1, 2 or 3, and    -   R₁ is selected among alkyl groups comprising from 1 to 10 carbon        atoms bearings at least one betaine group; and the electron        donor groups, notably O donors, S donors, NHCO donors, SCO        donors, NHCS donors, and SCS donors, said electron donors        bearing one or several betaine groups,        it being understood that when the group Ar2 represents a phenyl        group, it is substituted with at least one group R₁ including an        electron donor group.

In the complexes of formula (III) according to the invention, Ar2 is forexample, selected

-   -   c. either from the groups:

-   -   -    with Ar2 as defined for the complexes of formula (III)            (which corresponds to the case when L₁ represents a direct            or covalent bond),

    -   d. or from the groups:

-   -   -    with Ar2 which represents a phenyl, thiophenyl, furanyl,            pyrrolyl or imidazolyl group substituted with s groups R₁            either identical or different, s and R₁ being as defined for            the complexes of formula (III).

Preferably, in the complexes of formula (III) according to theinvention, the substituents R₁, either identical or different, areselected from:

-   -   L₂-Alk, -L₂-L₃-Q₁;    -   the NHCO donor groups are selected from: —NHCO(OAlk),        —NHCO(NHAlk), —NHCO(NAlk1Alk2), —NHCO(SAlk),    -   the SCO donor groups are selected from: —SCO(OAlk), —SCO(NHAlk),        —SCO(NAlk1Alk2), —SCO(SAlk),    -   the NHCS donor groups are selected from: —NHCS(OAlk),        —NHCS(NHAlk), —NHCS(NAlk1Alk2), and    -   the SCS donor groups are selected from: —SCS(OAlk), —SCS(NHAlk),        —SCS(NAlk1Alk2), —SCS(SAlk),    -   Alk, Alk1 and Alk2, either identical or different, are alkyl        groups comprising from 1 to 10 carbon atoms, substituted with at        least one betaine group,    -   Q₁ represents a betaine group or a branched group bearing at        least two betaine groups,    -   L₂ is a direct bond, —O—, —S—, —NHCO—, —SCO—, —NHCS— or —SCS—,    -   L₃ is a bond arm.

In the complexes of formula (III) and (IV) according to the invention,L₃ preferably represents a covalent bond, an alkylene group from 1 to 12carbon atoms, optionally comprising one or several double or triplebonds; a cycloalkylene group from 5 to 8 carbon atoms, an arylene groupfrom 6 to 14 carbon atoms; or a sequence of one or several alkylenegroups from 1 to 12 carbon atoms, cycloalkylene group from 5 to 8 carbonatoms and/or arylene group from 6 to 14 carbon atoms; said alkylene,cycloalkylene or arylene groups may comprise or not one or severalheteroatoms such as oxygen, nitrogen, sulphur, phosphorous or one orseveral carbamoyle or carboxamido groups and/or which may benon-substituted or substituted with one or several alkyl groups from 1to 8 carbon atoms, aryl from 6 to 14 carbon atoms, sulfonate or oxogroup; the following bond arms being preferred:

n is equal to 1, 2, 3, 4, 5 or 6,m, p and r, either identical or different, are equal to 1, 2 or 3.

When L₂ and L₃ each represent a direct bond (equally called a covalentbond), -L₂-L₃-Q₁, corresponds to -Q₁ and -L₂-L₃-Q₂ to -Q₂

In the complexes of formula (IV) and (III) defined within the scope ofthe invention, if Ln³⁺=Eu³⁺ or Snn³⁺, in this case, according to apreferred embodiment L₁ will be selected, which represents —C≡C—, whichleads to suitable luminescence properties.

In the complexes of formula (IV) and (III) defined within the scope ofthe invention, if Ln³⁺=Eu³⁺, Sm³⁺, Tb³⁺ or Dy³⁺ (and, in particular Tb³⁺or Dy³⁺), according to a preferred embodiment L₁ will be selected, whichrepresents a direct bond (also-called a covalent bond).

In the complexes of formula (IV) and (III) defined within the scope ofthe invention, the present betaine groups are for example zwitterionicgroups associating an ammonium cation, or aromatic iminium, generallypyridinium, imidazolium, and an anionic group of the sulfonate,phosphonate or carboxylate type, preferably sulfonate, the cation andthe anion being spaced apart by at least one ring member CH₂, andpreferably by a bivalent alkyl chain (also-called alkylene) comprisingfrom 1 to 4, or even from 1 to 6, carbon atoms. Preferably, the betainegroups present in the complexes of formula (IV) and (III) are selectedfrom:

with R which represents an alkyl group from 1 to 6 carbon atoms, andpreferably a methyl or ethyl, and q which is equal to 1, 2, 3, 4, 5 or6, and preferably which is equal to 1 or 2, the group —N(CH₃)₂⁺—(CH₂)₃—SO₃ ⁻ being preferred.

The object of the invention is also:

-   -   chelating agents of formula (II):

with Ra which is protective group of acid functions such as an alkyl, ofthe methyl type, and Z, Chrom1, Chrom2 and Chrom3, either identical ordifferent, which are as defined for the compounds of formula (IV); and

-   -   the ligands of formula (I):

with Ra which is a protective group of acid functions such as an alkyl,of the methyl type, and Chrom4 which is as defined for the compounds offormula (III).

Within the scope of the invention, by «alkyl» group, unless specifiedotherwise, is meant a linear or branched saturated hydrocarbon chain. Asan example of an alkyl group comprising from 1 to 6 carbon atoms,mention may notably be made of methyl, ethyl, n-propyl, iso-propyl,n-butyl, tert-butyl, sec-butyl, n-pentyl, n-hexyl groups.

By «cycloalkyl», is meant a saturated or unsaturated hydrocarbon groupconsisting of at least one ring, optionally a bridged ring. As anexample, mention may be made of cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, adamantyl and cyclohexene groups.

By «aryl» groups, are meant mono-, bi- or poly-cyclic groups, preferablyincluding from 6 to 12 carbon atoms, comprising at least one aromaticgroup, for example a phenyl, cinnamyl or naphthyl group, as well asheteroaryl groups. By heteroaryl group is meant a mono-, bi- orpoly-cyclic carbocycle, preferably including from 6 to 12 members, andcomprising at least one aromatic group and at least one heteroatomselected from oxygen, nitrogen or sulphur atom, integrated within thecarbocycle. As an example of a heteroaryl group, mention may be made of2-, 3- or 4-pyridinyl, 2- or 3-furoyl, 2- or 3-thiophenyl, pyrrolyl,imidazolyl, pyrazolyl, thiazolyl, oxazolyl, isoxazolyl, pyridinyl,pyrazinyl, pyrimidinyl, tetrazolyl, thiadiazolyl, oxadiazolyl,triazolyl, pyridazinyl, indolyl groups. Phenyl and 1,2,3-triazole areparticularly preferred aryl groups.

Advantageously, the complexes according to the invention areluminescent. The luminescence corresponds to a light emissionconsecutive to a supply of energy, in particular a supply of light. Thisenergy supply causes atoms or molecules to pass into an «excited» statelocated at a higher energy than that which they had in their normalstate, a so-called «fundamental» state. It is when they return to theirfundamental state that they may emit light. Generally, lanthanidecomplexes are sensitized by an antenna effect and the nature of theorganic antenna controls the absorption of light and allows optimizationof the luminescence of the lanthanide (S. V. Eliseeva, J.-C. G. Bünzli,Chem. Soc. Rev. 2010, 39, 189).

Within the scope of the invention, the antenna advantageously consistsof a pyridine derivative functionalized in position 4 by a conjugatesystem called a chromophore and notably defined under the names ofChrom1, Chrom2, Chrom3 and Chrom4 in the preferred complexes. Thischromophore optionally consists of a conjugate spacer selected fromdouble or triple carbon-carbon bonds, which advantageously correspondsto the —C≡C— bond, and of an aryl or heteroaryl group substituted with1, 2 or 3 groups, so that at least 2 betaine groups are present on thefinal complex. The antennas in which the aryl or heteroaryl group isdirectly bound to pyridine will be preferred for sensitizing europium,samarium, terbium and dysprosium complexes, while the antennas includinga conjugate spacer will be preferred for the europium and samariumcomplexes.

More particularly, the invention is applied to chelating ligandsdescribed in their protected form according to formula (I) or tochelating macrocycles described in their protected form according toformula (II) and to their corresponding lanthanide complexes accordingto formulae (III) and (IV).

According to formula (III) is described the family of lanthanidecomplexes from the coordination of 3 identical chelating ligands offormulae (I) after de-protection and release of the acid functions.

According to formula (IV), is described the family of lanthanidecomplexes from the coordination of a macrocyclic ligand of formula (II)after de-protection and release of the acid functions.

As an example of a sub-structure of the complexes of formula (III) and(IV) as exemplified in the examples, mention may be made of:

-   -   a) the complexes (IVa)

-   -    with L₁ as defined within the scope of the invention and which        preferably represents —C≡C—, Z as defined for the complexes of        formula (IV) and which preferably represents a carbon atom and        R₀ ¹, R₀ ² and R₀ ³, either identical or different, which        represent a group R₀ as defined for the complexes of formula        (IV), at least two of the groups R₀ ¹, R₀ ² and R₀ ³ being        substituted with at least one betaine group,    -   b) the complexes (IIIa)

-   -    with R₁ as defined for the complexes of formula (III).

The preparation of the complexes according to the invention appliesstandard techniques and reactions known to one skilled in the art. Theymay notably be obtained according to methods similar to those used inthe examples.

The starting reagents are commercially available or easily accessible.The compounds of formulae (I) and (II) are therefore chemicallyaccessible in a relatively standard way, and may be obtained at areasonable preparation cost.

The functional groups optionally present in the compounds of formula (I)and (II) and in the reaction intermediates may be protected during thesynthesis, either permanently or temporarily, with protective groupswhich ensure a one-one synthesis of the expected compounds. Theprotection and de-protection reactions are carried out according totechniques well known to one skilled in the art. By temporary protectivegroup of amines, alcohols or carboxylic acids, are meant the protectivegroups like those described in Protective Groups in Organic Synthesis,Greene T. W. and Wuts P. G. M., ed. John Wiley and Sons, 2006 and inProtecting Groups, Kocienski P. J., 1994, Georg Thieme Verlag.

The complexation reactions are generally conducted with a salt of thedesired lanthanide notably a chloride, a nitrate or a triflate in adissociating solvent, notably an alcohol and preferably methanol oracetonitrile, or further in a mixture of solvents, in the presence of abase, generally a carbonate, at a temperature comprised between 25 and80° C., for a period varying from 30 minutes to a few hours.

For example, in the case of complexes (IV), the complexing agents offormula (II) according to the invention, in the case when the threechromophores are identical (symmetrical complexing agents) may beprepared from the intermediate (A1):

wherein Ra is a protective group of acid functions such as an alkyl with1 to 4 carbon atoms, of the methyl type, Z is as defined for thecompounds of formula (IV) and Y which represents a chlorine, bromine andpreferably iodine atom, or a function —N₃ or —C≡CH.

The complexing agents of formula (II) according to the invention, in thecase when Chrom1=Chrom2≠Chrom3 (dissymmetrical complexing agents) may beprepared from the intermediate (A2):

wherein Ra is a protective group of acid functions such as an alkyl with1 to 4 carbon atoms, of the methyl type, Z is as defined for thecompounds of formula (IV) and Y which represents a chlorine, bromine, orpreferably iodine atom, or a function —N₃ or —C≡CH.

The introduction of groups Chrom1, Chrom2 and Chrom3 may be accomplishedin a single step in the case of symmetrical complexing agents from (A1)or in two steps in the case of dissymmetrical complexing agents from(A2) according to techniques detailed in the examples hereafter, oraccording to similar techniques well known to one skilled in the art.

Within the scope of the invention, the method applying a divergentsynthesis from the key intermediates (A1) and (A2) is more rapid andmore efficient for preparing complexing agents of formula (IV) than themethod proposed for preparing complexing agents in application WO2013/011236. In the synthesis method used within the scope of theinvention, the chromophores Chrom1, Chrom2 and Chrom3 or certain oftheir precursors are directly introduced on (A1) and (A2) by usingstandard carbon-carbon coupling reactions catalyzed with palladium, suchas the reaction of Sonogashira, of Heck, of Suzuki, of Stille, orfurther the dipolar 1,3 cycloaddition reactions catalyzed with copper,commonly called «click chemistry».

In every case, the chromophores Chrom1, Chrom2 and Chrom3 will befunctionalized with reactive functions compatible with the carbon-carbonor “click chemistry” coupling reactions which are envisioned. Thesefunctionalizations are conducted according to procedures from theliterature and known to one skilled in the art. Notably the presence ofterminal alkyne or alkene functions is notably contemplated in the caseof the chromophores Chrom1, Chrom2 and Chrom3 having a spacer,compatible with the Sonogashira and Heck reactions. The presence of aboronic acid function compatible with Suzuki reactions is alsocontemplated in the case of the chromophores Chrom1, Chrom2 and Chrom3without any spacer wherein the aryl or heteroaryl group is directlybound to pyridine.

Notably, the introduction of groups:

with Ar1, s and R₀ (it being understood that various groups R₀ maysubstitute the Ar1 group) which are as defined for the compounds offormula (IV), may be accomplished:

-   -   either by action of a compound of formula (V):

with R′₀ (it being understood that various groups R′₀ may substitute theAr1 group) which represents R₀ or a precursor group of R₀ and Ar1, s andR₀ are as defined for the compounds of formula (IV), on the intermediate(A1) or (A2), wherein Y is a chlorine, bromine or preferably iodineatom, under the conditions of Suzuki coupling,

-   -   or in the case when Ar1 represents a 1,2,3-triazole group, by        action of a compound of formula (VI):

with R′₀ which represents R₀ or a precursor group of R₀, R₀ being asdefined for the compounds of formula (IV), on the intermediate (A1) or(A2), wherein Y is N₃, by a so-called «click chemistry» reaction. It isalso possible to achieve coupling with a compound N₃—R′₀, the alkynefunction then being borne by the pyridine of the intermediate (A1) or(A2).

The introduction of groups:

with R₀ (it being understood that various groups R₀ may substitute thephenyl group) as defined for the compounds of formula (IV) may beaccomplished by action of a compound of formula (VII):

with R′₀ (it being understood that various groups R′₀ may substitute thephenyl group) which represents R₀ or a precursor group of R₀, and R₀ ands are as defined for the compounds of formula (IV), on the intermediate(A1) or (A2) wherein Y is a chlorine, bromine or preferably iodine atom,under the conditions of a Sonogashira reaction.

The complexes according to the invention of formula (III) are preparedfrom a ligand of type (I):

wherein Chrom4 is as defined earlier for the complexes of formula (III),and Ra is a protective group of acid functions such as an alkyl, forexample a methyl.

The complexing agents according to formula (I) are prepared in the sameway from intermediates of formula (A3):

wherein Ra is a protective group of acid functions such as an alkyl with1 to 4 carbon atoms, of the methyl type, and Y represents a chlorine,bromine, or preferably iodine atom, or a function —N₃ or —C≡CH and thesynthesis of which is described in A. Picot, C. Feuvrie, C. Barsu, B. LeGuennic, H. Le Bozec, C. Andraud, L. Toupet, O. Maury Tetrahedron. 2008,64, 399-411 and in Z. El Abidine Chamas, X. Guo, J.-L. Canet, A.Gautier, D. Boyer, R. Mahioub Dalton Trans., 2010, 39, 7091-7097.

Such ligands are prepared according to techniques detailed in theexamples hereafter, or according to similar techniques well known to oneskilled in the art. In particular, the introduction of groups:

with Ar2, s and R₁ (it being understood that various groups R₁ maysubstitute the Ar2 group) which are as defined for the compounds offormula (III), may be accomplished:

-   -   either by action of a compound of formula (VIII):

with R′₁ (it being understood that various groups R′₁ may substitute theAr2 group) which represents R₁ or a precursor group of R₁ and Ar2, s andR₁ are as defined for the compounds of formula (III), on the compound offormula (IX):

with X which is a chlorine, bromine or preferably iodine atom, and Rawhich is a protective group of acid functions such as an alkyl, of themethyl type, under the conditions of Suzuki coupling,

-   -   either in the case when Ar2 represents a 1,2,3-triazole group,        by action of a compound of formula:

with R′₁ which represents R₁ or a precursor group of R₁ and R₁ which isas defined for the compounds of formula (III), on the compound offormula (XI):

with Ra which is a protective group of acid functions such as an alkyl,of the methyl type, by a so-called click chemistry reaction. It is alsopossible to achieve the coupling with a compound N₃—R′₁, the alkynefunction then being borne by the pyridine of the ligands (I).

The introduction of groups:

with s and R₁ (it being understood that various groups R₁ may substitutethe phenyl group) as defined for the compounds of formula (III) may beensured by action of a compound of formula (XII):

with R′₁ (it being understood that various groups R′₁ may substitute thephenyl group) which represents R₁ or a precursor group of R₁, and R₁ ands are as defined for the compounds of formula (III), on the compound offormula (IX):

with X which is a chlorine, bromine or preferably iodine atom and Rawhich is a protective group of acid functions such as an alkyl, of themethyl type, under the conditions of a Sonogashira reaction.

Regardless of the contemplated synthesis route and the targetedlanthanide complex, the betaine groups may for example be introduced atthe substituents R₀ or R₁ via direct alkylation of a phenol, thiophenol,aniline group etc. . . . or else via a so called «click chemistry»reaction from a propargyl precursor.

In order to form the corresponding lanthanide complexes, the selectedligand (I) or the macrocycle of formula (II) is put into solution in asolvent in which it is soluble, for example ethanol or methanol, and thede-protection of the acid functions is achieved in a basic medium, andthen complexation is conducted with a salt of the desired rare earth,notably as a chloride, triflate or nitrate. Advantageously, about 3molar equivalents of ligand (I) are used per rare earth atom and about 1molar equivalent of macrocycle of formula (II) is used per atom of rareearth. The complexation may be achieved at a temperature from 25 to 80°C., for example for a period of the order of 30 minutes to 6 hours.

The examples hereafter allow an illustration of the invention, but donot have any limiting nature.

The acronyms below are used in the examples which follow:

-   ACN: Acetonitrile-   DMF: Dimethylformamide-   DPA: Dipicolinic acid-   Et₃N: Triethylamine-   MeOH: Methanol-   Ms: A mesyl group-   TA: Room temperature-   TACN: Triazacyclononane-   TBTA: Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine-   THF: Tetrahydrofurane-   TMSA: Trimethylsilylacetylene    General Information:

The NMR spectra (¹H and ¹³C) were recorded on a Bruker AC 200 apparatusat a frequency of 200.13 and 50.32 MHz for ¹H and ¹³C respectively andon a Bruker Advance at a frequency of 500.10 and 125.75 MHz for ¹H and¹³C respectively. The chemical shifts (δ) are expressed in parts permillion (ppm) relatively to the trimethylsilane used as an internalreference and by using the indicated solvents. The coupling constants(J) are expressed in Hz and the following notations are used: s(singlet), brs (broad singlet), d (doublet), dd (doublet of doublets), m(multiplet).

The high resolution mass spectra HRMS were recorded in the common centrefor mass spectrometry at Lyon(s) (Université Claude Bernard Lyon, Lyon,France) on a MicroTOFQII apparatus equipped with a positive ESI source.

The thin layer chromatographies were carried out on plates of silica gelon aluminium sheets (silica gel, Fluka) and revealed by means of a UVlamp (λ=254 or 365 nm) or by coloration.

The purifications were achieved with a chromatography column on silicagel (silica gel 0.035-0.070 mm, 60 A).

The solvents used for the reactions were purchased from Aldrich or AcrosOrganics as dry or extra dry solvents and stored on a 3 Å molecularsieve.

The UV/visible absorption spectra were recorded on a JASCO V670spectrometer; the emission spectra on a JOBIN-YVON fluorolog 3spectrofluorimeter.

A. PREPARATION OF THE COMPLEXES Example 1: Europium Complex of Formula(IV)

The intermediate 3 is obtained by an alkylation reaction of TACN.3HCl,from a mesylate (or tosylate) derivative under standard alkylationconditions. This compound will then be able to be engaged into variouscrossed coupling reactions in order to obtain the desired ligands.

The synthesis of the dissymmetrical intermediate 6 requires severalsteps for protection/de-protection of TACN and is prepared according tothe method described in patent WO 2013/011236.

TACN-boc 4 is substituted with the compound 2 in order to lead to thecompound 5, a precursor of the dissymmetrical intermediate 6 afterde-protection of the Boc function.

The synthesis of compound 10 may be achieved under standard substitutionconditions on 4-iodophenol from a commercial amine 8. The protectedalkyne function of the compound 10 may be introduced via a Sonogashiracrossed coupling reaction in the presence of TMSA. The introduction ofthe compound 10 on the intermediate 3 may be achieved afterde-protection of the alkyne, into a real alkyne, which is then directlyengaged into a modified Sonogashira coupling step (without CuI). Thebetaine function may be prepared by a reaction for opening1,3-propanesultone selectively on the terminal amine of the compound 11in order to lead to the complexing agent 12. The methyl esters may thenbe hydrolyzed and the obtained complexing agent reacts in situ in thepresence of a europium salt solution in order to lead to the complexEu2.

The compound 15 is obtained after substituting the compound 6 with theantenna 14. The compound 14 is obtained as described in patentapplication WO 2013/011236. The compound 16 may be obtained afterSonogashira coupling of the antenna 10b, as described earlier for thesymmetrical complex. Also, the compound 17 may be obtained after openingthe 1,3-propanesultone selectively on the terminal amines of compound16.

a) Preparation of Compound 2

The compound 1 (1.56 g, 5.33 mmol, 1.0 equiv.) is dissolved in 50 ml ofdistilled THF. Triethylamine (1.63 mg, 16 mmol, 3.0 equiv.) is thenadded, as well as mesyl chloride (976 mg, 8.53 mmol, 1.6 equiv.). Thesolution is stirred under argon at room temperature. The reaction istracked by TLC. After 4 h, the reaction is complete, the solvents areevaporated. The residue is solubilized in ethyl acetate and the organicphase is washed with a saturated NaHCO₃ solution, and then with water.The organic phase is then dried on Na₂SO₄, and then filtered. Thesolvent is removed under reduced pressure. The obtained raw product ispurified by a chromatography column on silica gel (Eluent:dichloromethane/methanol from 0% to 5% by volume, with increments of 1%)leading to the desired product as a white solid (1.40 g, 90%).

¹H NMR (500 MHz, CDCl₃) δ: 8.47 (d, ⁴J=1.5 Hz, 1H), 8.05 (d, ⁴J=1.5 Hz,1H), 5.37 (s, 2H), 4.00 (s, 3H), 3.16 (s, 3H)

¹³C NMR (500 MHz, CDCl₃) δ: 164, 155, 148, 134, 107, 70, 53, 38

HRMS (ESI⁺): (Calculated for C₉H₁₀NO₅SNa: 393.9212); measured: [M+Na]⁺:m/z=393.9217.

b) Preparation of Compound 3

To a suspension of Na₂CO₃ (1.59 g, 15 mmol, 10 equiv.) in 60 ml ofacetonitrile under argon are added TACN.3HCl (356 mg, 1.5 mmol, 1.0equiv.), and compound 2 (1.4 g, 4.79 mmol, 3.2 equiv.). The reactionmixture is stirred for 48 h at 60° C. under argon. After returning toroom temperature, the reaction mixture is filtered and the filtrate isevaporated under reduced pressure. The reaction crude is purified bychromatography on a column of neutral alumina (Eluent: ethyl acetate).The final product is obtained as a yellow solid (814 mg, 57%).

¹H NMR (200 MHz, CDCl₃) δ: 8.35 (d, J=1.4 Hz, 3H), 8.21 (d, J=1.4 Hz,3H), 3.97 (s, 9H), 3.93 (s, 6H), 2.90 (s, 12H)

¹³C NMR (125.76 MHz, CDCl₃) δ: 164.6, 161.9, 147.65, 147.62, 135.6,132.8, 106.65, 106.60, 63.9, 56.1, 53.2, 53.19, 53.14.

HRMS (ESI⁺): (Calculated for C₃₀H₃₄I₃N₆O₆: 954.9627); measured: [MH]⁺:m/z=954.9668.

c) Preparation of Compound 5

In a flask, the compound 4 (370 mg, 1.24 mmol, 1.0 equiv.) issolubilized in dry acetonitrile (100 ml). To this solution are addedunder argon Na₂CO₃ (789 mg, 7.44 mmol, 6.0 equiv.) and the compound 3(966 mg, 2.6 mmol, 2.1 equiv.). The reaction mixture is stirred underargon at 60° C. for 4 h. At the end of the reaction, the Na₂CO₃ isfiltered under reduced pressure and the solvents are evaporated. Thereaction crude is purified on a chromatography column on silica gel(Eluent: AcOEt). The product is obtained as a yellow oil (633 mg, 66%).

¹H NMR (500 MHz, CDCl₃) δ: 8.35 (2s, 2H), 8.24 (s, 1H), 8.13 (s, 1H),3.98 (2s, 2×3H), 3.95 (2s, 2×1H), 3.42 (brs, 2H), 3.36 (brs, 2H), 3.10(brs, 2H), 2.98 (brs, 2H), 2.7 (brs, 2H), 2.62 (brs, 2H), 1.48 (s, 9H).

¹³C NMR (125.76 MHz, CDCl₃) δ: 164.80, 164.73, 162.32, 162.13, 155.79,147.95, 147.77, 135.57, 135.39, 132.96, 132.92, 106.81, 106.76, 79.69,63.30, 63.03, 56.77, 55.15, 55.10, 54.59, 53.28, 50.94, 50.42, 28.91.

d) Preparation of Compound 6

In a flask, the compound 5 (50 mg, 6.41·10⁻² mmol, 1.0 equiv.) issolubilized in CH₂Cl₂ (2 ml). To this solution is added trifluoroaceticacid (20 equiv.). The reaction mixture is stirred under argon for 4 h.At the end of the reaction, the solvents are evaporated under reducedpressure. The product is obtained as a yellow oil and is directlyengaged into the next step without any other purification.

¹H NMR (500 MHz, CDCl₃) δ: 8.29 (s, 2H), 7.88 (s, 2H), 4.34 (brs, 4H),3.89 (s, 6H), 3.65 (brs, 4H), 3.51 (brs, 4H), 3.29 (brs, 4H).

e) Preparation of Compound 9

In a flask, the compound 7 (1.00 g, 4.54 mmol, 1.0 equiv.) issolubilized in anhydrous DMF (30 mL). To this solution are added theamine 8 (1.07 g, 6.81 mmol, 1.5 equiv.) and K₂CO₃ (6.30 g, 45.4 mmol, 10equiv.). The reaction mixture is stirred under argon at 80° C. for 12 h.After returning to room temperature, CH₂Cl₂ is added. The organic phaseis washed three times with an aqueous saturated NaCl solution, and thenwith water, dried on Na₂SO₄, filtered and evaporated. The product isobtained as a yellow powder without any other purification (1.20 g,87%).

¹H NMR (200 MHz, CDCl₃) δ: 7.54 (d, 2H, J=8.9 Hz), 6.73 (d, 2H, J=8.9Hz), 3.97 (t, 2H, J=6.4 Hz), 2.43 (t, 2H, J=6.9 Hz), 2.25 (s, 6H), 1.93(m, 2H).

¹³C NMR (50.32 MHz, CDCl₃) δ: 159.04, 138.26, 117.07, 82.65, 66.47,56.43, 45.67, 27.60.

HRMS (ESI⁺): (Calculated for C₁₁H₁₆INO: 305.0277); measured: [MH]⁺:m/z=306.0349

f) Preparation of Compound 10

In a Schlenk tube, the compound 9 (860 mg, 2.81 mmol, 1.0 equiv.) issolubilized in a THF/Et₃N mixture (20 ml, 1/1, v/v). The solution isdegassed under argon for 20 min and then TMSA (555 mg, 5.64 mmol, 2.0equiv.) is added, as well as the catalysts, PdCl₂(PPh₃)₂ (59 mg,7.86·10⁻⁵ mol, 0.03 equiv.) and CuI (50 mg, 2.64·10⁻⁴ mol, 0.1 equiv.).The reaction mixture is stirred under argon at room temperature for 16h. At the end of the reaction, the solvents are evaporated under reducedpressure. The reaction crude is dissolved in CH₂Cl₂ and the organicphase is washed with an aqueous solution saturated with NH₄Cl, and thenwith water. The organic phase is then dried on Na₂SO₄, filtered andevaporated under reduced pressure. The obtained crude product ispurified on a chromatography column on silica gel. Eluent CH₂Cl₂/MeOH(from 0 to 5% by volume). The desired compound is obtained as an orangeoil (650 mg, 84%).

¹H NMR (200 MHz, CDCl₃) δ: 7.36 (d, 2H, J=8.9 Hz), 6.78 (d, 2H, J=8.9Hz), 3.97 (t, 2H, J=6.4 Hz), 2.43 (t, 2H, J=7.3 Hz), 2.24 (s, 6H), 1.93(m, 2H), 0.22 (s, 9H).

¹³C NMR (50.32 MHz, CDCl₃) δ: 159.33, 133.54, 115.21, 115.21, 114.46,105.42, 92.39, 66.32, 56.42, 45.66, 27.61.

HRMS (ESI⁺): (Calculated for C₁₆H₂₅NOSi: 275.1705); measured: [MH]⁺:m/z=276.1805

g) Preparation of Compound 10b

In a flask, the compound 10 (445 mg, 1.61 mmol, 1.0 equiv.) issolubilized in anhydrous MeOH (15 ml). To this solution is added K₂CO₃(268 mg, 1.93 mmol, 1.2 equiv.). The reaction mixture is stirred at roomtemperature for one hour. At the end of the reaction, the solvent isevaporated under reduced pressure. The residue is solubilized in CH₂Cl₂and the organic phase is washed twice with water, and then dried onNa₂SO₄, filtered and evaporated under reduced pressure. The compound isobtained as a brown oil and is directly engaged into the next stepwithout any other purification.

¹H NMR (200 MHz, CDCl₃) δ: 7.36 (d, 2H, J=8.9 Hz), 6.84 (d, 2H, J=8.9Hz), 4.02 (t, 2H, J=6.5 Hz), 2.99 (s, 1H), 2.44 (t, 2H, J=7.0 Hz), 2.25(s, 6H), 1.95 (m, 2H).

h) Preparation of Compound 11

In a Schlenk tube, the derivative 3 (64 mg, 6.76·10⁻⁵ mol, 1.0 equiv.)is solubilized in a DMF/Et₃N mixture (2 ml, 1/1, v/v). The solution isdegassed under argon for 30 min. To this solution are then added asolution of 10b (55 mg, 2.70·10⁻⁴ mol, 4.0 equiv.) solubilized in DMF (1ml) degassed beforehand under argon, and then PdCl₂(PPh₃)₂ (4 mg,5.68·10⁻⁶ mmol, 0.03 equiv.). The reaction mixture is stirred underargon for 48 h at 80° C. At the end of the reaction, the solvents areevaporated under reduced pressure. CH₂Cl₂ is added to the reaction crudeand the organic phase is washed with a saturated Na₂CO₃ solution, andthen with water. The organic phase is then dried on Na₂SO₄, filtered andthen evaporated under reduced pressure. The reaction crude is purifiedby successive precipitations in an AcOEt/pentane mixture in order tolead to the desired compound as an orange oil (52 mg, 65%).

¹H NMR (500 MHz, CDCl₃) δ: 8.03 (s, 3H), 7.85 (s, 3H), 7.45 (d, 6H,J=8.8 Hz), 6.88 (d, 6H, J=8.8 Hz), 4.03 (t, 6H, J=6.2 Hz), 3.96 (m,15H), 2.96 (brs, 12H), 2.45 (t, 6H, J=6.4 Hz), 2.26 (s, 18H), 1.97 (m,6H).

¹³C NMR (125.76 MHz, CDCl₃) δ: 165.73, 161.57, 160.12, 147.47, 133.76,133.60, 128.74, 127.95, 125.60, 114.87, 113.93, 95.56, 85.65, 66.47,64.47, 56.40, 56.16, 53.13, 45.59, 45.41, 27.49.

HRMS (ESI⁺): (Calculated for C₆₉H₈₄N₉O₉: 394.2142); measured: [M+3H]³⁺:m/z=394.2125

i) Preparation of Compound 12:

The compound 11 (36 mg, 3.05·10⁻⁵ mol, 1.0 equiv.) is solubilized in 1ml of acetone. 1,3-propanesultone is added and the reaction mixture isstirred under argon at room temperature for 16 h. At the end of thereaction, the solvent is evaporated and the residue is triturated inCH₂Cl₂. The product is obtained as an orange oil (40 mg, 85%).

¹H NMR (200 MHz, CDCl₃) δ: 7.83 (s, 3H), 7.67 (s, 3H), 7.38 (d, 6H,J=8.9 Hz), 6.93 (d, 6H, J=8.9 Hz), 4.26 (brs, 4H), 4.16-4.10 (m, 8H),3.92 (s, 9H), 3.69-3.48 (m, 16H), 3.17 (brs, 12H), 2.94-2.90 (m, 6H),2.37-2.13 (m, 6H)

j) Preparation of the Complex 13, Eu2

The compound 12 (25 mg, 1.65·10⁻⁵ mol, 1.0 equiv.) is solubilized in aMeOH/H₂O mixture (4 ml, 1/1, v/v). To this solution is added an aqueousNaOH solution (1M) (4 ml) and the reaction mixture is stirred underargon for one hour. At the end of the reaction an aqueous solution ofHCl is added down to a pH=2. To this mixture are then added a solutionof Na₂CO₃ (8.0 equiv.) as well as the salt EuCl₃.6H₂O (3.0 equiv.). Thereaction mixture is stirred under argon at 50° C. for 16 h. At the endof the reaction, the solvents are evaporated under reduced pressure andthe complex is purified by dialysis in H₂O for 24 h. The product isobtained as a white powder.

HRMS (ESI⁺): (Calculated for C₇₅H₉₀EuN₉Na₄O₁₈S₃: 436.3593); measured:[M+4Na]⁴⁺: m/z=436.3589

k) Preparation of Compound 15

The compound 6 (86 mg, 1.28·10⁻⁴ mol, 1.0 equiv.) is solubilized in 4 mlof ACN. To this solution is added under argon the compound 14 (86 mg,1.66·10⁻⁴ mol, 1.3 equiv.) and Na₂CO₃ (40 mg, 3.84·10⁻⁴ mol, 3.0equiv.). The reaction mixture is stirred under argon at 50° C. for 24 h.At the end of the reaction, the solvents are evaporated and the crudeproduct is purified by chromatography column on Al₂O₃ (Eluent:CH₂Cl₂/MeOH from 0 to 5% of MeOH). The product is obtained as a yellowoil (m=60 mg, 42%)

¹H NMR (300 MHz, CDCl₃) δ: 8.07-8.06 (m, 2H), 7.98 (s, 1H), 7.79 (s,1H), 7.62 (s, 1H), 7.20 (d, 2H, J=8.5 Hz), 6.62 (d, 2H, J=8.5 Hz),3.81-3.76 (m, 4H), 3.73-3.64 (m, 13H), 3.09-3.03 (m, 4H), 2.74-2.62 (m,9H), 1.73 (t, 2H, J=6.37 Hz), 1.17 (s, 9H).

Example 2: The Terbium Complex of Formula (IV)

In the same way, the intermediate 3 may react under Suzuki couplingconditions in the presence of boronic acid 18 in order to lead to theligand 19. The methyl esters are then hydrolyzed in a basic medium andthe compound reacts in situ in the presence of Tb(III) salt in order tolead to the formation of the complex 20.

The complex Tb2 bearing betaine functions may be obtained via the samesynthesis route as its analogue 20. The intermediate 3 may react via aSuzuki reaction with the corresponding antenna 21. The ligand 22 mayreact in the presence of 1,3-propanesultone in order to lead to theligand 23. As previously, the complex will be formed in situ in thepresence of the corresponding Tb(III) salt in order to lead to thecomplex Tb2.

a) Preparation of Compound 19:

In a Schlenk tube, the intermediate 3 (150 mg, 1.57·10⁻⁴ mol, 1.0equiv.) and boronic acid 18 (79 mg, 5.18·10⁻⁴ mol, 3.3 equiv.) aresolubilized in anhydrous DMF (4 ml). The solution is degassed underargon for 30 min. To this solution are then added Cs₂CO₃ (215 mg,6.59·10⁻⁴ mol, 4.2 equiv.) and Pd(PPh₃)₄ (10 mg, 9.42·10⁻⁶ mmol, 0.06equiv.). The reaction mixture is stirred under argon for 72 h at 100° C.At the end of the reaction, Cs₂CO₃ is filtered under reduced pressureand rinsed several times with CH₂Cl₂. The filtrate is evaporated underreduced pressure and diluted in CH₂Cl₂. The organic phase is washed witha saturated NaCl solution, and then with water. The organic phase isthen dried on Na₂SO₄, filtered and then evaporated under reducedpressure. The reaction crude product is purified by successiveprecipitations from an AcOEt/pentane mixture in order to lead to thedesired compound as an orange oil (58 mg, 41%).

¹H NMR (200 MHz, CDCl₃) δ: 8.19 (s, 3H), 8.02 (s, 3H), 7.58 (d, 6H,J=8.8 Hz), 6.94 (d, 6H, J=8.9 Hz), 4.02 (s, 6H), 3.99 (s, 9H), 3.85 (s,9H), 3.02 (brs, 12H).

¹³C NMR (50.32 MHz, CDCl₃) δ: 166.33, 161.79, 161.03, 149.37, 147.95,129.60, 128.35, 123.33, 121.21, 114.81, 65.05, 56.48, 55.59, 29.90.

HRMS (ESI⁺): (Calculated for C₅₁H₅₅N₆O₉: 895.4032); measured: [MH]⁺:m/z=895.4025.

b) Preparation of the Complex Tb2

The Tb2 complex is prepared in a similar way to the complex Eu2, byusing the procedure described in Example 1, paragraph f).

HRMS (ESI⁺): (Calculated for C₄₈H₄₅N₆Na₂O₉Tb: 527.1144); measured:[M+2Na]²⁺: m/z=527.1143

Example 3: Terbium Complex of Formula (III)

In the case of complexes of DPA derivatives, the introduction of betainegroups may be achieved via two different synthesis routes. The betainegroup may be introduced in a single “click chemistry” step, asillustrated in Scheme 6, in the case of precursors 24 and 25 for leadingto the ligand 26. The methyl esters are then hydrolyzed in a basicmedium and the intermediate reacts in the presence of Tb(III) salts forleading to the complex Tb1.

a) Preparation of Compound 26

The derivative 24 (300 mg, 1.27 mmol, 1.0 equiv.) and the compound 25(315 mg, 1.52 mmol, 1.2 equiv.) are solubilized in THF (5 ml). To thissolution is added a freshly prepared solution of a mixture of CuSO₄.5H₂O(6.5 mg, 0.02 mmol, 0.02 equiv.), of TBTA (15 mg, 0.07 mmol, 0.06equiv.) and of sodium ascorbate (14 mg, 0.02 mmol, 0.02 equiv.) in aTHF/H₂O mixture. The reaction mixture is stirred for 16 h at roomtemperature. The solvents are then evaporated and the crude product istriturated in acetone. The formed precipitate is filtered under reducedpressure and the desired product is obtained as a white powder (400 mg,72%).

¹H NMR (500 MHz, D₂O) δ: 9.20 (s, 1H), 8.82 (s, 2H), 4.83 (s, 2H), 4.03(s, 6H), 3.53-3.50 (m, 2H), 3.18 (s, 6H), 2.97 (t, 2H, J=6.8 Hz),2.38-2.34 (m, 2H).

¹³C NMR (125.76 MHz, D₂O) δ: 166.09, 150.81, 146.44, 137.82, 128.60,120.08, 63.40, 58.78, 54.89, 51.64, 48.35, 19.49.

HRMS (ESI⁺): (Calculated for C₁₇H₂₄N₅O₇S: 442.1318); measured: [M]⁺:m/z=442.1391; [M-H+Na]⁺: m/z=464.1214.

b) Preparation of the Complex Tb1

The complex Tb1 is prepared in a similar way to the complex Eu2, byusing the procedure described in Example 1, paragraph f), in thepresence of 3.0 equivalents of ligand 18.

Example 4: Europium Complex of Formula (IV)

The precursor 27 may be obtained via a Sonogashira reaction on thecompound DPA-I. The betaine function is in this case introduced byopening the 1,3-propanesultone on the terminal dimethylamine. The methylesters of the intermediate 28 may then be hydrolyzed in a basic mediumand the formed intermediate reacts in the presence of a Eu(III) salt inorder to lead to the complex Eu1.

a) Preparation of Compound 27

In a Schlenk tube, the compound DPA-I (215 mg, 6.72·10⁻⁴ mol, 1.0equiv.) is solubilized in a THF/Et₃N mixture (7 ml, 1/1, v/v). Thesolution is degassed under argon for 20 min and then the compound 10b(265 mg, 1.30 mmol, 1.2 equiv.) is added, as well as the catalysts,PdCl₂(PPh₃)₂ (5 mg, 2.0·10⁻⁵ mol, 0.03 equiv.) and CuI (5 g, 6.72·10⁻⁴mol, 0.1 equiv.). The reaction mixture is stirred under argon at roomtemperature for 16 h. At the end of the reaction, the solvents areevaporated. The reaction crude product is dissolved in CH₂Cl₂ and theorganic phase is washed with an aqueous saturated NH₄Cl solution, andthen with water. The organic phase is then dried on Na₂SO₄, filtered andevaporated under reduced pressure. The obtained crude is purified by achromatography column on silica gel. Eluent CH₂Cl₂/MeOH (from 0 to 5%).The desired compound is obtained as a yellow powder (210 mg, 80%).

¹H NMR (200 MHz, CDCl₃) δ: 8.31 (s, 2H), 7.48 (d, 2H, J=8.9 Hz), 6.90(d, 2H, J=8.9 Hz), 4.90-3.97 (m, 6+2H), 2.51 (t, 2H, J=HZ), 2.33 (s,6H), 2.03 (m, 2H).

¹³C NMR (50.32 MHz, CDCl₃) δ: 164.81, 16.26, 148.34, 134.92, 133.80,129.45, 114.80, 113.27, 97.59, 84.61, 66.17, 56.16, 53.31, 45.21, 27.03.

HRMS (ESI⁺): (Calculated for C₂₂H₂₅N₂O₅: 397.1744); measured: [MH]⁺:m/z=397.1758

b) Preparation of Compound 28:

The compound 27 (70 mg, 1.76·10⁻⁴ mol, 1 equiv.) is solubilized inanhydrous acetone. To this solution is added under argon1,3-propanesultone (22 mg, 1.76·10⁻⁴ mol, 1 equiv.). The reactionmixture is stirred under argon for 2 h. At the end of the reaction, theformed precipitate is filtered under reduced pressure and triturated inan Acetone/CH₂Cl₂ mixture (1/1, v/v) and then again filtered. Thedesired product is obtained as a yellow powder (80 mg, 88%)

¹H NMR (500 MHz, MeOD) δ: 8.27 (s, 2H), 7.57 (d, 2H, J=8.9 Hz), 7.01 (d,2H, J=8.9 Hz), 4.16 (t, 2H, J=6.0 Hz), 4.01 (s, 6H), 3.60-3.57 (m, 4H),3.17 (s, 6H) 2.89 (t, 2H, J=6.4 Hz), 2.34-2.30 (m, 2H), 2.27-2.23 (m,2H).

¹³C NMR (125.76 MHz, MeOD) δ: 166.03, 161.40, 149.98, 136.21, 135.16,130.34, 116.15, 98.18, 85.60, 79.63, 66.03, 63.96, 62.97, 53.65, 30.82,23.93, 20.06

HRMS (ESI⁺): (Calculated for C₂₅H₃₀N₂NaO₈S: 541.1615); measured:[M+Na]⁺: m/z=541.1615

c) Preparation of Complex Tb1:

The Tb1 complex is prepared similarly to the complex Eu2, by using theprocedure described in Example 1, paragraph f), in the presence of 3.0equivalents of ligand 28.

B. EVALUATION OF THE SPECTROSCOPIC PROPERTIES OF THE COMPLEXES

a) Spectroscopic Properties of the Complex Eu2 in Water.

The Eu2 complex has good solubility in water of the order of 10⁻⁴-10⁻⁵mol·L⁻¹), as well as excellent stability in water down to dilutions ofthe order of 10⁻⁷ mol·L⁻¹. The single FIGURE shows A. the absorptionspectrum of the complex Eu2 in water; B. the emission spectrum of theEu2 complex recorded in water (▪_(excit)=310 nm).

The complex Eu2 has remarkable spectroscopic properties in waterλ_(max)=335 nm, ε=50,000 L·mol⁻¹·cm⁻¹; Φ=0.18 and τ=0.8 ms, as well as asignificant two photon cross-section. Its luminosity at 337 nm istherefore estimated to be 9,000 L·mol⁻¹·cm⁻¹, in the same order ofmagnitude as its homologues functionalized with PEG fragments describedin application WO 2013/011236. These results show that the introductionof betaine functions is not detrimental to the spectroscopic propertiesof the complex.

In the case of the other synthesized complexes, it is also noticed thatby introducing betaine it is possible to improve the solubility of thecomplexes (as compared with analogue complexes without any betaine) anddoes not significantly modify the luminescent properties of the complex.

C. STUDY IN A CELL MEDIUM

The compound Eu2 allowed images to be made of bound cells by single ordouble photon microscopy. Cancer cells T24 (bladder cancer cell lineATCC, HTB-4, see A. Picot, A. D'Aléo, P. L. Baldeck, A. Grichine, A.Duperray, C. Andraud, O. Maury J. Am. Hem. Soc. 2008, 130, 1532), weresent with methanol and put into the presence of a solution of thecomplex (final concentration in the medium=10⁻⁵ mol·L⁻¹). The images ofthe cells were recorded on a confocal Zeiss microscope operating undersingle or double photon conditions.

It was observed that the complex Eu2 reversibly labeled the cell.Indeed, after washing, the complex disappears from the cell clearlyindicating that functionalization with betaines does not causeirreversible accumulation in lipophilic tissues, underlying forcesobserved when the PEG groups are used. This clearly shows that thepresence of the three betaine groups gives the possibility of avoidingnon-specific interactions with the lipophilic portions of the cell.

The invention claimed is:
 1. Lanthanide complexes selected amonglanthanide complexes of formula (IV):

wherein: Ln is a lanthanide, chosen among Eu, Sm, Tb or Dy, Z represents—C— or —PR₃—, R₃ represents a phenyl, benzyl, methyl, ethyl, propyl,n-butyl, sec-butyl, iso-butyl or tert-butyl group, Chrom1, Chrom2 andChrom3, either identical or different, are selected from the groups:

wherein: L₁ represents a direct bond, —C═C— or —C≡C—, Ar₁ represents anaromatic group selected among phenyl, thiophenyl, furanyl, pyrrolyl,imidazolyl or triazolyl groups, substituted with s R₀ groups, eitheridentical or different, s is equal to 1, 2 or 3, and R₀ is selectedfrom: alkyl groups comprising from 1 to 10 carbon atoms bearing at leastone betaine function and/or one reactive function; and electron donorgroups selected from O donors, S donors, NHCO donors, SCO donors, NHCSdonors, and SCS donors, said electron donor groups may either bear ornot bear one or several betaine groups, and/or a reactive function,wherein, when all the groups Ar1 represent a phenyl group, at least oneof these phenyl groups is substituted with at least one R₀ groupincluding an electron donor group; wherein at least two of the groupsChrom1, Chrom2 and Chrom3 are substituted with at least one R₀ groupbearing at least one betaine group.
 2. The lanthanide complexesaccording to claim 1, having, at most, 12 betaine groups.
 3. Thelanthanide complexes according to claim 1, wherein the at least onereactive function is selected from —COOH, —NH₂, an acrylamide, anactivated amine, an activated ester, an aldehyde, an alkyl halide, ananhydride, an aniline, an azide, an aziridine, a carboxylic acid, adiazoalkane, a haloacetamide, a halotriazine, a hydrazine, an imidoester, an isocyanate, an isothiocyanate, a maleimide, a sulfonyl halide,a thiol, a ketone, an amine, an acid halide, a hydroxysuccinimidylester, a succinimidyl ester, a hydroxysulfosuccinimidyl ester, anazidonitrophenyl, an azidophenyl, a 3-(2-pyridyl-dithio)-propionamide, aglyoxal, a triazine, an acetylene group, and groups of formula:

wherein w is an integer belonging to the range from 0 to 8 and v isequal to 0 or 1, and Ar is a saturated or unsaturated heterocycle with 5or 6 members, comprising from 1 to 3 heteroatoms, optionally substitutedwith a halogen atom.
 4. The lanthanide complexes according to claim 1,wherein Chrom1, Chrom2 and Chrom3 are defined as follows: a. eitherChrom1, Chrom2 and Chrom3, either identical or different, are selectedfrom the groups:

b. or Chrom1, Chrom2 and Chrom3, either identical or different, areselected from the groups:

with Ar1 which represents a phenyl, thiophenyl, furanyl, pyrrolyl orimidazolyl group substituted with s R₀ groups, either identical ordifferent.
 5. The lanthanide complexes according to claim 1, wherein thesubstituents R₀, either identical or different, are selected among:-L₂-Alk, -L₂-L₃-Q₁ and -L₂-L₃-Q₂; the NHCO donor groups selected among:—NHCO(OAlk), —NHCO(NHAlk), —NHCO(NAlk1Alk2), —NHCO(SAlk), the SCO donorgroups selected among: —SCO(OAlk), —SCO(NHAlk), —SCO(NAlk1Alk2),—SCO(SAlk), the NHCS donor groups selected among: —NHCS(OAlk),—NHCS(NHAlk), —NHCS(NAlk1Alk2), and the SCS donor groups selected among:—SCS(OAlk), —SCS(NHAlk), —SCS(NAlk1Alk2), —SCS(SAlk), Alk, Alk1 andAlk2, either identical or different, are alkyl groups comprising from 1to 10 carbon atoms, optionally substituted with at least one betainegroup, Q₁ represents a betaine group or a branched group bearing atleast two betaine groups, L₂ is a direct bond, —O—, —S—, —NHCO—, —SCO—,—NHCS— or —SCS—, L₃ is a linker, and Q₂ is a reactive group able toallow a covalent bonding with a molecule of interest to be marked,wherein at least two of the present R₀ substituents bear at least onebetaine group, so that at least two of the groups Chrom1, Chrom2 andChrom3 are substituted with at least one R₀ group bearing at least onebetaine group.
 6. The lanthanide complexes according to claim 1, whereinChrom1=Chrom2=Chrom3.
 7. The lanthanide complexes according to claim 1,wherein Chrom1=Chrom2 and are substituted with at least one R₀ groupbearing at least one betaine group and Chrom3 is substituted with atleast one R₀ group bearing a function -L₂-L₃-Q₂, wherein Q₂ represents agroup selected among —COOH, —NH₂, an acrylamide, an activated amine, anactivated ester, an aldehyde, an alkyl halide, an anhydride, an aniline,an azide, an aziridine, a carboxylic acid, a diazoalkane, ahaloacetamide, a halotriazine, a hydrazine, an imido ester, anisocyanate, an isothiocyanate, a maleimide, a sulfonyl halide, a thiol,a ketone, an amine, an acid halide, a hydroxysuccinimidyl ester, asuccinimidyl ester, a hydroxysulfosuccinimidyl ester, anazidonitrophenyl, an azidophenyl, an 3-(2-pyridyl-dithio)-propionamide,a glyoxal, a triazine, an acetylene group, and the groups of formula:

wherein w is an integer belonging to the range from 0 to 8 and v isequal to 0 or 1, and Ar is a saturated or unsaturated heterocycle with 5or 6 members, comprising 1 to 3 heteroatoms, optionally substituted witha halogen atom.
 8. The lanthanide complexes according to claim 1,wherein L₁ represents a direct bond or —C≡C—, and the groups Ar1, eitheridentical or different, each represent a phenyl group, substituted withs R₀ groups, either identical or different.
 9. The lanthanide complexesaccording to claim 1, wherein, in all the groups Ar1, s is equal to 1.10. The lanthanide complexes according to claim 1, wherein the groupsAr1, either identical or different, each represent a phenyl groupselected from the groups:


11. The lanthanide complexes according to claim 1, selected among thelanthanide complexes of formula (III):

wherein Ln is a lanthanide, chosen among Eu, Sm, Tb or Dy, Chrom4 isselected from the groups:

wherein: L₁ represents a direct bond, —C═C— or —C≡C—, Ar2 represents anaromatic group selected among the phenyl, thiophenyl, furanyl, pyrrolyl,imidazolyl or triazolyl groups, said groups Ar2 being substituted with sR₁ groups, either identical or different, s is equal to 1, 2 or 3, andR₁ is selected among alkyl groups comprising from 1 to 10 carbon atomsbearing at least one betaine group; and electron donor groups selectedfrom O donors, S donors, NHCO donors, SCO donors, NHCS donors, and SCSdonors, said electron donor groups bearing one or several betainegroups, wherein, when the group Ar2 represents a phenyl group, it issubstituted with at least one R₁ group including an electron donorgroup.
 12. The lanthanide complexes according to claim 11, wherein Ar2is selected: a. either from the groups:

b. or from the groups:

with Ar2 which represents a phenyl, thiophenyl, furanyl, pyrrolyl orimidazolyl group substituted with s R₁ groups, either identical ordifferent.
 13. The lanthanide complexes according to claim 1, whereinthe substituents Ru either identical or different, are selected among:-L₂-Alk, -L₂-L₃-Q₁; the NHCO donor groups selected among: —NHCO(OAlk),—NHCO(NHAlk), —NHCO(NAlk1Alk2), —NHCO(SAlk), the SCO donor groupsselected among: —SCO(OAlk), —SCO(NHAlk), —SCO(NAlk1Alk2), —SCO(SAlk),the NHCS donor groups selected among: —NHCS(OAlk), —NHCS(NHAlk),—NHCS(NAlk1Alk2), and the SCS donor groups selected among: —SCS(OAlk),—SCS(NHAlk), —SCS(NAlk1Alk2), —SCS(SAlk), Alk, Alk1 and Alk2, eitheridentical or different, are alkyl groups comprising from 1 to 10 carbonatoms, substituted with at least one betaine group, Q₁ represents abetaine group or a branched group bearing at least two betaine groups,L₂ is a direct bond, —O—, —S—, —NHCO—, —SCO—, —NHCS— or —SCS—, L₃represents a bond arm selected among a covalent bond, an alkylene groupfrom 1 to 12 carbon atoms, optionally comprising one or several doubleor triple bonds; a cycloalkylene group from 5 to 8 carbon atoms, anarylene group from 6 to 14 carbon atoms; or a sequence of one or severalalkylene groups from 1 to 12 carbon atoms, cycloalkylene groups from 5to 8 carbon atoms and/or arylene groups from 6 to 14 carbon atoms; saidalkylene, cycloalkylene or arylene groups may comprise one or severalhetero-atoms or not such as oxygen, nitrogen, sulfur, phosphorus atomsor one or several carbamoyl or carboxamido groups and/or may benon-substituted or unsubstituted with one or several alkyl groups from 1to 8 carbon atoms, aryl groups from 6 to 14 carbon atoms, sulfonate oroxo groups.
 14. The lanthanide complexes according to claim 13, whereinL₃ is selected from:

n is equal to 1, 2, 3, 4, 5 or 6, m, p and r, either identical ordifferent, are equal to 1, 2 or
 3. 15. The lanthanide complexesaccording to claim 1, wherein: either Ln³⁺=Eu³⁺ or Sm³⁺ and L₁represents —C≡C—; or Ln³⁺=Eu³⁺, Sm³⁺, Tb³⁺ or Dy³⁺, and L₁ represents adirect bond.
 16. The lanthanide complexes according to claim 1, whereinthe betaine groups are selected from zwitterionic groups associating anammonium or aromatic iminium cation selected among pyridinium,imidazolium cation, and an anionic group selected among sulfonate,phosphonate or carboxylate groups, said cation and said anion beingspaced apart by at least one CH₂.
 17. The lanthanide complexes accordingto claim 1, wherein the betaine groups are selected among:

with R which represents an alkyl group from 1 to 6 carbon atoms, and qis equal to 1, 2, 3, 4, 5 or
 6. 18. The lanthanide complexes accordingto claim 1, wherein the reactive function is selected from —COOH, —NH₂,succinimidyl esters, haloacetamides, azides, hydrazines, isocyanates,and maleimides.
 19. The lanthanide complexes according to claim 1,wherein the betaine group is —N(CH₃)₂ ⁺—(CH₂)₃—SO₃ ⁻.
 20. The lanthanidecomplexes according to claim 7, wherein Q₂ is selected among —COOH,—NH₂, succinimidyl esters, haloacetamides, hydrazines, isocyanates, andmaleimides.
 21. The lanthanide complexes according to claim 15, whereinLn³⁺=Tb³⁺ or Dy³⁺.